Visualized Detection of Tobacco Anthracnose by a Recombinase Polymerase Amplification–Lateral Flow Dipstick Assay

Anthracnose caused by <i>Colletotrichum</i> spp. is a widespread fungal disease that is detrimental to tobacco growth and inflicts economic damage of up to 100 million in tobacco-growing regions in China. An early diagnostic tool is vital for the accurate determination and management of anthracnose in the field. This study investigated the diversity of <i>Colletotrichum</i> spp. on tobacco leaves with anthracnose and developed a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) diagnostic method for the rapid and equipment-independent detection of the main <i>Colletotrichum</i> spp. causing tobacco anthracnose. This assay targeted the chitin synthase gene (<i>chs1</i>) and could be performed in a few minutes (6 to 10 min). All isolates of <i>C</i>. <i>karstii</i>, <i>C</i>. <i>fructicola</i>, and <i>C</i>. <i>gloeosporioides</i> yielded positive results in the RPA-LFD assay, and no cross-reaction occurred with other fungal species from tobacco or other hosts. The detection threshold was 1 pg of genomic DNA under optimal reaction conditions. The entire RPA-LFD assay enabled the detection of pathogen visualization within 30 min without specialized equipment by combining a polyethylene glycol-KOH method for extracting gDNA rapidly from tobacco leaves infected with <i>C</i>. <i>karstii</i>, <i>C</i>. <i>fructicola</i>, and <i>C</i>. <i>gloeosporioides</i>. Based on these results, the RPA-LFD assay is easy to operate, rapid, and equipment-independent and is promising for development as a kit to diagnose tobacco anthracnose in resource-limited settings at point-of-care.